PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD) are devastating neurological diseases with no effective therapies. Mutation in the C9orf72 gene is the most common genetic cause of ALS and FTD (c9FTD/ALS). The C9orf72 gene contains a polymorphic hexanucleotide repeat, GGGGCC, located in an intron, which in unaffected individuals is typically between 5 and 10 repeats but expands to hundreds or thousands in ALS. The repeat-containing mRNAs have splicing defects and are translated cytoplasmically through a mechanism called repeat associated non-AUG (RAN) translation to make toxic dipeptide repeat proteins. The mechanism of RAN translation, and how RNA structure and exogenous factors dictate non-canonical initiation and elongation through the repeats remains unresolved. Our preliminary data show that (1) RAN translation occurs through a cap-dependent scanning mechanism, (2) RAN translation occurs in distinct reading frames, leading to different dipeptide repeats, and (3), there are specific genetic modulators of RAN translation. Most importantly, through an unbiased genetic screen we discovered deletion of ribosomal protein RPS25, a known modulator of non-canonical translation initiation, specifically inhibits RAN translation in yeast and human cells, and is neuroprotective in animal models expressing C9orf72 repeat expansions. Here we build on these data, combining genetic approaches and expertise in ALS (Gitler) with structural and biophysical approaches to translation (Puglisi) to delineate the mechanism of RAN translation in 3 specific aims. In Aim 1, we will use biochemical and single- molecule methods to determine the mechanistic pathways of RAN translation initiation and elongation, and delineate the role of repeat expansion mRNA structure, likely formed by G- quadruplexes, in start site selection during initiation, and subsequent reading frame selection and maintenance during elongation. We will apply cryoEM and other structural methods to describe stable intermediates identified in this aim. In Aim 2, we will use these methods to understand the specific role of RPS25 and other factors identified by genetic screens for inhibitors or enhancers of RAN translation. We will focus on RPS25, using bulk and single- molecule methods, coupled with structural/biochemical analyses on RPS25 knockout ribosomes. In Aim 3, we will use genetic screens to identify further modifiers of RAN translation, and characterize further those identified by our past screens, validating them across yeast, human and animal models. Our results should illuminate how RAN translation leads to neurotoxic protein production, providing potential therapeutic targets for FTD/ALS.